Among retroviruses, lentiviruses such as HIV-1 display two distinct virological features: genomic hypervariation and infection of nondividing/terminally differentiated cells (i.e. macrophage). These traits contribute to viral evolution and pathogenesis, respectively. Our recent research suggests that these two unique virological characteristics of HIV-1 are mechanistically related and require a novel enzymatic property of the viral reverse transcriptase (RT): high binding affinity to dNTP substrates. During the previous funding period of this award, we identified a unique enzymatic property of HIV-1 RT, compared to MuLV RT. Indeed, we found that HIV-1 RT binds to the dNTP substrate with much higher affinity than MuLV RT. Interestingly, due to this unique dNTP binding property, HIV-1 RT efficiently catalyzes both DNA synthesis and mismatch extension (which contributes to its low enzyme fidelity), even at low dNTP concentration. We also found that two HIV-1 RT mutants with reduced dNTP binding affinity, V148I and Q151N, show reduced DNA synthesis efficiency at low dNTP concentrations and increased fidelity. Indeed, HIV-1 variants harboring these mutant RTs failed to infect macrophage containing low dNTP concentrations even though these viruses retain their ability to infect dividing cells containing high dNTP concentrations. In addition, due to their low dNTP binding affinity, these mutants display frequent stalling and elevated RNA template switching even at physiological dNTP concentrations. In fact, the Q151N mutant virus showed the highest viral recombination frequency among all RT mutants ever tested. The central hypothesis of this proposal is that the relatively high dNTP binding affinity of lentiviral RTs may be essential for two of the most important (and seemingly unrelated) features of the lentivirus family: their abilities to infect terminally differentiated cells and to undergo high rates of genomic mutation. First, we will perform a series of biochemical and virological experiments to elucidate the unique structural and mechanistic architecture of the HIV-1 RT active site, which is responsible for its high dNTP binding affinity. In this aim, both currently available HIV-1 RT mutants with altered dNTP binding affinity and in vivo HIV-1 RT variants that have been isolated from the HIV-1 infected patients will be employed. Second, we will test whether mutations of HIV-1 RT reducing dNTP binding affinity also reduces proviral DNA synthesis kinetics at low cellular dNTP concentrations, leading to loss of viral infectivity to macrophages. Third, we will test the effect of dNTP binding affinity of HIV-1 RT on the viral recombination frequency. This proposed work is expected to provide insight into the impact of HIV-1 RT on a range of key biological features of HIV- 1: cell tropism, genomic mutability, recombination and pathogenesis.